Methods in molecular biology
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Antisense oligonucleotide induced exon skipping emerges as a promising therapeutic strategy for patients suffering from a devastating muscle disorder Duchenne muscular dystrophy (DMD). Systemic administration of antisense phosphorodiamidate morpholino oligomers (PMOs) targeting exons 6 and 8 in dystrophin mRNA of the canine X-linked muscular dystrophy model in Japan (CXMDJ) that lacks exon 7, restored dystrophin expression throughout skeletal muscle and ameliorated skeletal muscle pathology and function. However, the antisense PMO regime used in CXMDJ could not be considered for a direct application to DMD patients so far, because this type of mutation is quite rare. ⋯ The accompanying skipping of exon 9, which does not alter the reading frame, varied according to the cell origin. The antisense PMOs originally administered to the CXMDJ dog model were capable of inducing multi-exon skipping of the dystrophin gene on the FACS-aided MyoD-transduced fibroblasts derived from an exon 7-deleted DMD patient. These data support the suitability of dog as a laboratory model for DMD because the similarity of dystrophin sequences allowed a successful translation of the dog's PMOs to DMD patients cells.
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The PI3K/AKT/mTOR signaling pathway shows frequent molecular alterations and increased activity in cancer. Given its role in the regulation of cell growth, survival and metastasis, molecules within this pathway are promising targets for pharmacologic intervention. ⋯ In this article, we summarize results from preclinical studies and clinical trials that examined PI3K pathway inhibitors in BLCA focusing on technical challenges that might result in contradictory findings in preclinical studies. Based on published data from our group, we also address challenges that need to be overcome to optimize PI3K inhibition in BLCA and enable its successful translation into the clinic.
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Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide. It is a silently growing epidemic with multifaceted pathogenesis, and current standards of treatments aim to target only the symptoms of the primary injury, while there is a tremendous need to explore interventions that can halt the progression of the secondary injuries. The use of a reliable animal model to study and understand the various aspects the pathobiology of TBI is extremely important in therapeutic drug development against TBI-associated complications. ⋯ The present method describes how the CCI model could be utilized for determining the BBB dysfunction and hyperpermeability associated with TBI. Blood-brain barrier disruption is a hallmark feature of the secondary injury that occur following TBI, frequently associated with leakage of fluid and proteins into the extravascular space leading to vasogenic edema and elevation of intracranial pressure. The method described here focuses on the development of a CCI-based mouse model of TBI followed by the evaluation of BBB integrity and permeability by intravital microscopy as well as Evans Blue extravasation assay.
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Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach for the treatment of various genetic diseases and a therapy which has gained significant traction in recent years following FDA approval of new antisense-based drugs. Exon skipping for Duchenne muscular dystrophy (DMD) works by modulating dystrophin pre-mRNA splicing, preventing incorporation of frame-disrupting exons into the final mRNA product while maintaining the open reading frame, to produce a shortened-yet-functional protein as seen in milder Becker muscular dystrophy (BMD) patients. Exons 45-55 skipping in dystrophin is potentially applicable to approximately 47% of DMD patients because many mutations occur within this "mutation hotspot." In addition, patients naturally harboring a dystrophin exons 45-55 in-frame deletion mutation have an asymptomatic or exceptionally mild phenotype compared to shorter in-frame deletion mutations in this region. ⋯ In the case of DMD, researchers have often relied upon human muscle fibers obtained from muscle biopsies for testing; however, this method is highly invasive and patient myofibers can display limited proliferative ability. To overcome these challenges, researchers can generate myofibers from patient fibroblast cells by transducing the cells with a viral vector containing MyoD, a myogenic regulatory factor. Here, we describe a methodology for assessing dystrophin exons 45-55 multiple skipping efficiency using antisense oligonucleotides in human muscle cells derived from DMD patient fibroblast cells.
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Exon skipping is a therapeutic approach that is feasible for various genetic diseases and has been studied and developed for over two decades. This approach uses antisense oligonucleotides (AON) to modify the splicing of pre-mRNA to correct the mutation responsible for a disease, or to suppress a particular gene expression, as in allergic diseases. Antisense-mediated exon skipping is most extensively studied in Duchenne muscular dystrophy (DMD) and has developed from in vitro proof-of-concept studies to clinical trials targeting various single exons such as exon 45 (casimersen), exon 53 (NS-065/NCNP-01, golodirsen), and exon 51 (eteplirsen). ⋯ Permanent exon skipping achieved at the DNA level using clustered regularly interspaced short palindromic repeats (CRISPR) technology holds promise in current preclinical trials for DMD. In hopes of achieving clinical success parallel to DMD, exon skipping and splice modulation are also being studied in other muscular dystrophies, such as Fukuyama congenital muscular dystrophy (FCMD), dysferlinopathy including limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM), and distal anterior compartment myopathy (DMAT), myotonic dystrophy, and merosin-deficient congenital muscular dystrophy type 1A (MDC1A). This chapter also summarizes the development of antisense-mediated exon skipping therapy in diseases such as Usher syndrome, dystrophic epidermolysis bullosa, fibrodysplasia ossificans progressiva (FOP), and allergic diseases.